Methods of Eliciting Broadly Neutralizing Antibodies Targeting HIV-1 GP41

ABSTRACT

The present invention is directed to the induction and characterization of a humoral immune response targeting “entry-relevant” gp41 structures. In its broadest aspect, the present invention is directed to methods of raising a neutralizing antibody response to a broad spectrum of HIV strains and isolates. The present invention targets particular molecular conformations or structures that occur at the cell surface of HIV during viral entry into host cells. Such a humoral response can be generated in vivo as a prophylactic measure in individuals to reduce or inhibit the ability of HIV to infect uninfected cells in the individual&#39;s body. Such a response can also be employed to raise antibodies against “entry relevant” gp41 structures. These antibodies can be employed for therapeutic uses, and as tools for further illuminating the mechanism of HIV cell entry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit under 35 U.S.C. §119(d)(e) toU.S. Provisional Application No. 60/115,404, filed Jan. 8, 1999, theentire contents of which is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention pursuant to INNOVATION Grant No. R21 AI 42714.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to HIV therapy and prophylaxis. Inparticular, the invention relates to methods for eliciting broadlyneutralizing antibodies that target entry-relevant structures of HIV-1gp41. Such methods, and pharmaceutical compositions therefor, can beemployed to inhibit HIV entry into uninfected cells.

2. Related Art

The development of effective vaccines to prevent infection with HIVremains a high priority goal. To date, envelope glycoproteins (gp160 andgp120/gp41) have been the main focus of vaccine research efforts. Oneresult of this work is the observation that the humoral responsegenerated against native forms of the envelope (primarily oligomericforms of the gp120/gp41 complex) is more broadly neutralizing thanantibody raised against denatured and/or monomeric envelope (VanCott, T.C., et al., J. Virol. 71:4319-4330 (1997)). Structural considerationsare important components for both understanding the immunogenicity ofthe envelope protein and the design of envelope based immunogens whichinduce a broad neutralizing response against HIV.

A good deal of structural information is available with respect to thetransmembrane protein (TM or gp41). Predictive work indicated thatseveral regions of the ectodomain of gp41 display a high propensity toexhibit certain specific types of secondary structure (Gallaher, W. R.,et al., AIDS Res. Hum. Retroviruses 5:431-440 (1989); Delwart, E. L., etal., AIDS Res. Hum. Retroviruses 6:703-704 (1990)). Experimental workemploying both synthetic peptides and protein recombinants hasestablished that these predictions were generally correct and recently athree dimensional structure for a portion of the gp41 ectodomain wasreported (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541(1992); Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:12676-12680(1994); Wild, C., et al., AIDS Res. Hum. Retroviruses 11:323-325 (1995);Chan, D. C., et al, Cell 89:263-273 (1997)). Results from both solutionstudies and crystallographic analysis indicate that in one form thisstructured region of the transmembrane protein is a trimer of twointeracting regions of gp41. This trimeric structure is a six helixbundle consisting of an interior parallel coiled-coil trimer (regionone) which associates with three identical α-helices (region two) whichpack in an oblique, antiparallel manner into the hydrophobic grooves onthe surface of the coiled-coil trimer (FIG. 3). This hydrophobicself-assembly domain is believed to constitute the core structure ofgp41.

A series of studies carried out using both synthetic peptides andrecombinant proteins modeling the distal regions of the TM involved ingenerating this structure suggest that it (or the gp41 regions fromwhich it is derived) plays a critical role in the process of HIV-1 entry(Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992);Wild, C., et al., AIDS Res. Hum. Retroviruses 9:1051-1053 (1993); Wild,C., et al., Proc. Natl. Acad. Sci. USA 91:12676-12680 (1994); Wild, C.,et al., AIDS Res. Hum. Retroviruses 11:323-325 (1995); Wild, C., et al.,Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994); Chen, C.-H., et al., J.Virol. 69:3771-3777 (1995)).

The functional role of the transmembrane protein of HIV-1 in virusreplication was shown when the region of the ectodomain of the TMcorresponding to amino acid residues 558-595, which was predictive ofα-helical secondary structure (Gallaher, W. R., et al., AIDS Res. Hum.Retroviruses 5:431-440 (1989); Delwart, E. L., et al., AIDS Res. Hum.Retroviruses 6:703-704 (1990)), formed a coiled-coil structure whenmodeled as a synthetic peptide (Wild, C., et al., Proc. Natl. Acad. Sci.USA 89:10537-10541(1992)). The peptide modeling this region, DP-107, wasshown to be a potent, virus specific inhibitor of HIV replication andthe inhibitory activity was related to the structural componentsexhibited by the peptide. In both neutralization and cell-cell fusionassays, the DP-107 peptide completely blocked virus infection atconcentrations of 1.0 μg/ml. Unlike other inhibitors of HIV replication(i.e. soluble CD4) and most neutralizing sera, the activity of theDP-107 peptide was not isolate restricted. Using a series of DP-107analogs containing structure disrupting point mutations and a set ofHIV-1 envelope constructs containing identical mutations, it has beenshown that the structural components of the coiled-coil region of the TMwere critical to both virus entry and fusion phenotype and thatmutations which disrupted this gp41 structure gave rise to an envelopecomplex which was unable to mediate virus entry (Wild, C., et al., Proc.Natl. Acad. Sci. USA 91:12676-12680 (1994)).

Studies of the coiled-coil domain of gp41 resulted in the identificationof a second region of the ectodomain of the TM, which when modeled as asynthetic peptide, was also a potent, virus specific inhibitor of HIVreplication (Wild, C., et al., AIDS Res. Hum. Retroviruses 9:1051-1053(1993)). However, unlike the DP-107 region, the peptide corresponding toamino acid residues 643-678 of the TM (DP-178), did not exhibit stablesolution structure. Experiments with the DP-107 and DP-178 peptidesestablished that both of these materials blocked HIV replication at anearly step, most likely during virus entry (Wild, C., et al., Proc.Natl. Acad. Sci. USA 91:9770-9774 (1994)). This observation led tospeculation that these peptides might inhibit virus replication byinteracting with and disrupting determinants within the TM that werecritical for virus entry. Efforts to better define the higher orderstructural components that were present in gp41 and functioned duringvirus entry led to the observation that the distal regions of the TMmodeled by the two inhibitory peptides (DP-107 and DP-178) did interactwith one another to form an oligomeric structure (Wild, C., et al., AIDSRes. Hum. Retroviruses 11:323-325 (1995); Chen, C.-H., et al., J. Virol.69:3771-3777 (1995)). Recently, this oligomeric structure wascharacterized as a trimeric, six helix bundle consisting of an interiorparallel coiled-coil trimer (DP-107 region) which associates with threeidentical α-helices (DP-178 region) which pack into the hydrophobicgrooves on the surface of the coiled-coil trimer (FIG. 3) (Chan, D. C.,et al, Cell 89:263-273 (1997)).

Research has focused on determining the functional role of these gp41structural determinants in virus entry. DP-107 and DP-178 peptidesinteract in a specific manner with the ectodomain of gp41 and thisinteraction is critical to their inhibitory activities.

U.S. Pat. No. 5,464,933, Bolognesi et al., describes peptides whichexhibit potent anti-retroviral activity. Specifically disclosed are thepeptide DP-178 (SEQ ID NO:3) derived from the HIV-1_(LAI) gp41 protein,as well as fragments, analogs and homologs of DP-178. The peptides areused as direct inhibitors of human and non-human retroviral transmissionto uninfected cells. The patent teaches that the peptides may also beprophylactically employed in individuals after such individuals have hadan acute exposure to HIV.

U.S. Pat. No. 5,656,410, Wild et al., describes protein fragmentsderived from the HIV transmembrane glycoprotein (gp41), including thepeptide DP-107 (SEQ. ID NO:1) which have antiviral activity. Alsodisclosed are methods for inhibiting enveloped viral infection, andmethods that modulate biochemical processes involving coiled coilpeptide interactions.

While recent work has increased knowledge of the structural componentsof the HIV-1 transmembrane protein, the immunogenic nature of gp41remains poorly understood. It is known that one of two immunodominantregions present in the HIV-1 envelope complex is located in gp41 (Xu,J.-Y., et al., J. Virol. 65:4832-4838 (1991)). This determinant (TMresidues 597-613) is associated with a strong, albeit non-neutralizinghumoral response in a large number of HIV+ individuals. Also, thebroadly neutralizing antibody, 2F5, maps to the ectodomain of gp41 (TMresidues 662-667) (Muster, T., et al., J. Virol. 67:6642-6647 (1993);Muster, T., et al., J. Virol. 68:4031-4034 (1994)). It is interesting tonote that this antibody maps to a determinant of the TM that overlapsone of the two regions of gp41 which interact to form the recentlycharacterized hydrophobic core of the protein (FIG. 1). This observationhas lead to speculation that 2F5 might actually neutralize virus byinteracting with and disrupting the function of an entry-relevant gp41structure. An extensive study which mapped the antigenic structure ofgp41 supports this idea. This work characterized several conformationdependent gp41 MAbs which mapped to the same region of the TM as 2F5(Earl, P. L., et al., J. Virol. 71:2647-2684 (1997)). Although thebinding sites for these non-neutralizing monoclonal antibodies (MAbs)overlapped the 2F5 determinant, in competition experiments neither ofthese antibodies was blocked from binding to native protein by the 2F5MAb. This indicates that while the two dimensional regions to whichthese antibodies map are similar, the three dimensional epitopes towhich they bind are quite different.

The observation that only one neutralizing MAb (2F5) maps to theectodomain of gp41 and that antibodies to the 2F5 epitope are poorlyrepresented in sera from HIV infected individuals suggests that, for themost part, gp41 neutralizing epitopes are cryptic. The cryptic nature ofthese neutralizing epitopes is most likely related to the functionalrole of the TM in HIV-1 replication which involves mediating virusentry. It has been shown that prior to gp120-CD4 binding the HIVenvelope complex exists in a non-fusogenic form. While the exact natureof this pre-entry form is unknown, binding experiments have establishedthat the non-fusogenic state is characterized by the inaccessibility oflarge portions of the gp41 ectodomain (Sattentau, Q. J. and J. P. Moore,J. Exp. Med. 174:407-415 (1991); Sattentau, Q. J., et al., Virol.206:713-717 (1995)). However, once binding of virus to target cell hasoccurred, the gp120-gp41 complex undergoes a series of conformationalchanges that involve reorganization of both the extracellular surfacecomponent of the HIV-1 envelope protein (SU or gp120) and TM proteinsand the formation of structural components within the TM which arebelieved to be critical to virus entry. Although the steps involved inthe transition from the) non-fusogenic to fusogenic state are largelyunknown, it is believed that this transformation is characterized by theformation of a series of structural intermediates within thetransmembrane protein which drive the conformational changes requiredfor virus entry. The transitory nature of this event and the structuresassociated with it, rather than the absence of appropriate structuraldeterminants, are believed to account for the poor neutralizing responseto the TM component of the envelope system.

Attention has been given to the development of vaccines for thetreatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120,gp41) have been shown to be the major antigens for anti-HIV antibodiespresent in AIDS patients (Barin, et al., Science 228:1094-1096 (1985)).Thus far, these proteins seem to be the most promising candidates to actas antigens for anti-HIV vaccine development. To this end, severalgroups have begun to use various portions of gp160, gp120, and/or gp41as immunogenic targets for the host immune system. However, prior artattempts have thus far met with minimal success.

Thus, although a great deal of effort is being directed to the designand testing of HIV vaccines, an effective vaccine is needed.

SUMMARY OF THE INVENTION

An objective of the present invention is the induction and/orcharacterization of a humoral immune response targeting “entry-relevant”gp41 structures. In its broadest aspect, the present invention isdirected to methods of raising a neutralizing antibody response to abroad spectrum of HIV strains and isolates. The present inventiontargets particular molecular conformations or structures that occur, orare exposed, following interaction of HIV with the cell surface duringviral entry. Such a humoral response can be generated in vivo as aprophylactic or therapeutic measure in individuals to reduce or inhibitthe ability of HIV to infect uninfected cells in the individual's body.Such a response can also be employed to raise antibodies against “entryrelevant” gp41 structures. These antibodies can be subsequently employedfor therapeutic uses, and as tools for further illuminating themechanism of HIV cell entry.

One aspect of the present invention relates to a method of raising abroadly neutralizing antibody response to HIV by administering to amammal a peptide or polypeptide comprising an amino acid sequence thatis capable of forming a stable coiled-coil solution structurecorresponding to or mimicking the heptad repeat region of gp41, (or theN-helical domain of gp41). Peptides of this aspect of the invention areexemplified by P-15 and P-17 described herein.

A second aspect of the present invention relates to a method of raisinga broadly neutralizing antibody response to HIV by administering to amammal a peptide or polypeptide comprising an amino acid sequence thatcorresponds to, or mimics, the transmembrane-proximal amphipathicα-helical segment of gp41 (at the C-helical domain of gp41), or aportion thereof. Peptides of this aspect of the invention areexemplified by P-16 and P-18 described herein.

A third aspect of the present invention relates to a method of raising abroadly neutralizing antibody response to HIV by administering to amammal a composition including one or more peptides or polypeptideswhich comprise amino acid sequences that are capable of forming solutionstable structures that correspond to, or mimic, the gp41 core six helixbundle. This bundle forms in gp41 by the interaction of the distalregions (N-helical domain and C-helical domain) of the transmembraneprotein. See FIG. 1. This aspect of the invention is also directed tonovel mixtures of peptides and polypeptides, including multimeric andconjugate structures, wherein said mixtures and structures form a stablecore helix solution structure. A preferred embodiment of this aspect ofthe invention involves raising antibodies to a physical mixture ofN-helical domain peptide and C-helical domain peptide, for example, P-17and P-18, P-15 and P-16, P-17 and P-16, or P-15 and P-18.

The present invention is also directed to a method of raising a broadlyneutralizing antibody response to HIV by administering to a mammal acomposition including one or more novel peptides and proteins, hereinreferred to as conjugates, that mimic fusion-active transmembraneprotein structures. These conjugates are formed from two or more aminoacid sequences that comprise:

-   -   (a) one or more amino acid sequences that are capable of forming        a stable coiled-coil solution structure corresponding to or        mimicking the heptad repeat region of gp41 (N-helical domain);        and    -   (b) one or more amino acid sequences that correspond to, or        mimic, an amino acid sequence of the transmembrane-proximal        amphipathic α-helical segment of gp41 (C-helical domain);        wherein

said one or more sequences (a) and (b) are alternately linked to oneanother via a bond, such as a peptide bond (amide linkage) or by anamino acid linking sequence consisting of about 2 to about 25 aminoacids. These conjugates are preferably recombinantly produced. Anexample of such a conjugate is described in Example 5.

In a preferred embodiment of this aspect of the invention, one or moreof these conjugates folds and assembles in solution into a structurecorresponding to, or mimicking, the gp41 core six helix bundle.

The present invention also relates to methods for forming peptides,multimers and conjugates of the invention.

The present invention also relates to pharmaceutical compositionscomprising the peptides, multimers and conjugates of the invention and apharmaceutical acceptable carrier.

The present invention also relates to polyclonal and monoclonalantibodies that are raised to the peptides, multimers and conjugatesdescribed in the preceding paragraphs.

The present invention also relates to a method of administering acomposition comprising polyclonal or monoclonal antibodies describedabove to an individual in an amount effective to reduce HIV infection ofuninfected cells.

The present invention also relates to a vaccine for providing aprotective response in an animal comprising one or more peptides,multimers or conjugates of the present invention together with apharmaceutically acceptable diluent, carrier, or excipient, wherein thevaccine may be administered in an amount effective to elicit an immuneresponse in an animal to HIV. In a preferred embodiment, the animal is amammal. In another preferred embodiment, the mammal is a human.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the structural and antigenic regions of HIV-1 gp41.The extracellular, transmembrane and cytoplasmic domains are shown, asare the transmembrane-proximal amphipathic α-helical segment of gp41(C-helical domain) and the heptad repeat region of gp41 (N-helicaldomain).

FIG. 2 illustrates the formation of multimeric peptide constructscorresponding to the heptad repeat region of gp41 (represented by P-17)and one or more suitable linker peptides.

FIG. 3 illustrates the construction of conjugates of the inventionderived from repeating gp 41 fragments; and their subsequent folding andinteraction to form immunologically relevant epitopes.

FIG. 4 depicts the analysis of polyclonal sera to various immunogens bysurface immunoprecipitation. The precipitations were performed in thepresence (+) or absence (−) of 10 μg/ml sCD4.

FIG. 5 depicts analysis of polyclonal sera to various immunogens inneutralization assays. Immune sera or pre-immune (prebleed) sera werediluted 1:10 and incubated with various concentrations of virus(indicated in numbers of tissue culture infectious doses—TCID50). Levelsof virus replication were measured by the amount of p24 in thesupernatant seven days following infection, and normalized to the degreeof replication in the absence of any rabbit serum. The positive (+ve)control used is a strongly neutralizing serum from an HIV-1 infectedindividual.

FIG. 6: Percent neutralization for gp233 and gp234 sera in differentexperimental formats. FIG. 6 a shows the titration of bleed 2 for eachanimal against HIV-1_(MN) in the cell killing assay which uses cellviability as a measure of virus neutralization. MT2 cells are added to amixture of virus (sufficient to result in greater than 80% cell death at5 days post infection) and sera which had been allowed to incubate forapproximately 1 hr. After 5 days in culture, cell viability is measuredby vital dye metabolism. FIG. 6 b shows the percent neutralization foreach bleed at a 1:10 dilution against HIV-1_(MN) in an assay formatemploying CEM targets and p24 endpoint. In this assay, sera areincubated with 200 TCID₅₀ of virus for 1 hr prior to the addition ofcells. On days 1, 3, and 5 media are changed. On day 7 culturesupernatants are collected and analyzed for virus replication by p24antigen levels. In each assay format, percent neutralization isdetermined by comparison of experimental wells with cell and cell/viruscontrols.

FIG. 7 provides an example of a construct of the present invention (SEQ.ID NO:75) along with the corresponding nucleic acid sequence used forrecombinant expression of the construct (SEQ. ID NO:76).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The transitory-nature of the HIV-entry event, and the structuresassociated with it, account for the seeming lack of neutralizingepitopes within gp41. These structural components, which form andfunction only during virus entry, and remain unexposed or are notpresent in the “native” fusion-inactive envelope complex, constitute anovel set of neutralizing epitopes within gp41. The present inventioninvolves immunization with constructs mimicking these highly conserved,gp41 structures involved in virus entry to elicit the production ofbroadly neutralizing antibodies targeting these structures. Thus, thisinvention is the induction of a humoral immune response targeting these“entry relevant” gp41 structures.

One aspect of the present invention relates to a method of raising abroadly neutralizing antibody response to HIV by administering to amammal a peptide or polypeptide comprising an amino acid sequence thatis capable of forming a stable coiled-coil solution structurecorresponding to or mimicking the heptad repeat region of gp41 which islocated in the N-helical domain as defined herein. Peptides, ormultimers thereof, that comprise amino acid sequences which correspondto or mimic solution conformation of the heptad repeat region of gp41can be employed in this aspect of the invention. The heptad repeatregion of gp41 includes 4 heptad repeats. Preferably, the peptidescomprise about 28 to 55 amino acids of the heptad repeat region of theextracellular domain of HIV gp41 (N-helical domain, (SEQ. ID NO:1)), ormultimers thereof. The peptides can be administered as a small peptide,or conjugated to a larger carrier protein such as keyhole limpethemocyanin (KLH), ovalbumin, bovine serum albumin (BSA) or tetanustoxoid.

Alternatively, peptides forming a stable coiled-coil solution structurecorresponding to or mimicking the heptad repeat region of gp41 can beemployed to form polyclonal or monoclonal antibodies that can besubsequently administered as therapeutic or prophylactic agents.

To determine whether a particular peptide or multimer will possess astable trimeric coiled-coil solution structure corresponding to ormimicking the heptad repeat region of gp41, the peptide can be testedaccording to the methods described in Wild, C., et al., Proc. Natl.Acad. Sci. USA 89:10537-10541(1992), fully incorporated by referenceherein.

Shown below is the sequence for residues of the HIV-1_(LAI) gp41 proteinthat form the N-helical domain of the protein:

(SEQ. ID NO: 1) ARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKD QQLLGI

Two examples of useful peptides include the peptide P-17, which has theformula, from amino terminus to carboxy terminus, of:

(SEQ ID NO: 2) NH₂-NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ-COOH;and the peptide P-15, which has the formula, from amino terminus tocarboxy terminus, of:

(SEQ ID NO: 3) NH₂-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-COOH.These peptides are optionally coupled to a larger carrier protein, oroptionally include a terminal protecting group at the N- and/orC-termini. Useful peptides further include peptides corresponding toP-17 or P-15 that include one or more, preferably 1 to 10 conservativesubstitutions, as described below. A number of additional usefulN-helical region peptides are described in the section entitled“Peptides.”

A second aspect of the present invention relates to a method of raisinga broadly neutralizing antibody response to HIV by administering to amammal a peptide or polypeptide comprising an amino acid sequence thatcorresponds to, or mimics, the transmembrane-proximal amphipathicα-helical segment of gp41 (C-helical domain, (SEQ ID NO:4)), or aportion thereof. Useful peptides or polypeptides include an amino acidsequence that is capable of forming a core six helix bundle when mixedwith a peptide corresponding to the heptad repeat region of gp41, suchas the peptide P-17. Peptides can be tested for the ability to form acore six helix bundle employing the system and conditions described inChan, D. C., et al, Cell 89:263-273 (1997); Lu, M., et al., NatureStruct. Biol. 2:1075-1082 (1995), fully incorporated by referenceherein.

Shown below is the amino acid sequence for residues of the HIV-1_(LAI)gp41 protein that form the C-helical domain of the protein:

(SEQ ID NO: 4) WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN WFNITNW

Preferred peptides or multimers thereof, that can be employed in thisaspect of the invention comprise about 6 or more amino acids, preferablyabout 24-56 amino acids, of the extracellular C-helical domain of HIVgp41. The peptides can be administered as a small peptide, or conjugatedto a larger carrier protein such as keyhole limpet hemocyanin (KLH),ovalbumin, bovine serum albumin (BSA) or tetanus toxoid. Thistransmembrane-proximal amphipathic α-helical segment is exemplified bythe peptides P-16 and P-18, described below.

Alternatively, peptides or polypeptides comprising amino acid sequencesthat correspond to, or mimic, the transmembrane-proximal amphipathicα-helical segment of gp41, or a portion thereof, can be employed to formpolyclonal or monoclonal antibodies as therapeutic or prophylacticagents.

Examples of useful peptides for this aspect of the invention include thepeptide P-18 which corresponds to a portion of the transmembrane proteingp41 from the HIV-1_(LAI) isolate, and has the 36 amino acid sequence(reading from amino to carboxy terminus):

(SEQ ID NO: 5) NH₂-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-COOH;and the peptide P-16, which has the following amino acid sequence(reading from amino to carboxy terminus):

(SEQ ID NO: 6) NH₂-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-COOHThese peptides are optionally coupled to a larger carrier protein.Useful peptides further include peptides corresponding to P-18 or P-16that include one or more, preferably 1 to 10 conservative substitutions,as described below. In addition to the full-length P-18, 36-mer and thefull length P-16, the peptides of this aspect of the invention mayinclude truncations of the P-18 and P-16, as long as the truncations iscapable of forming a six helix bundle when mixed with P-17. A number ofother useful peptides are described in the section entitled “Peptides,”below.

A third aspect of the present invention relates to a method of raising abroadly neutralizing antibody response to HIV by administering to amammal a composition including one or more peptides or polypeptideswhich comprise amino acid sequences that are capable of forming solutionstable structures that correspond to, or mimic, the gp41 core six helixbundle. This bundle forms in gp41 by the interaction of the distalregions of the transmembrane protein, the heptad repeat region and theamphipathic α-helical region segment roughly corresponding to theN-helical domain and C-helical domain. See FIG. 1. The bundle structuresthat form in native virus are the result of a trimeric interactionbetween three copies each of the heptad repeat region and thetransmembrane-proximal amphipathic α-helical segment. In thecompositions of the present invention, peptide regions interact with oneanother to form a core six helix bundle. This aspect of the invention isalso directed to novel mixtures of peptides and polypeptides, includingmultimeric and conjugate structures, wherein said structures form astable core helix solution structure.

This aspect of the invention can employ mixtures of (a) one or morepeptides that comprise an amino acid sequence that corresponds to, ormimics, a stable coiled coil heptad repeat region of gp41; and (b) oneor more peptides that comprise a region that corresponds to, or mimics,the transmembrane-proximal amphipathic α-helical segment of gp41. Thesemixtures are optionally chemically or oxidatively cross-linked toprovide additional immunogenic structures that may or may not besolution stable. In addition to physical mixtures, and conventionalcross-linking, the peptides (a) and (b) can be conjugated together viasuitable linking groups, preferably a peptide residue having at least 2,preferably 2 to 25, amino acid residues. Preferred linking groups areformed from combinations of glycine and serine, or combinations ofglycine and cysteine when further oxidative cross-linking is envisioned.

A preferred embodiment of this aspect of the invention involves raisingantibodies to physical mixtures of P-17 and P-18, P-15 and P-16, P-17and P-16 or P-15 and P-18.

The present invention is also directed to a method of raising a broadlyneutralizing antibody response to HIV by administering to a mammal acomposition including one or more novel peptides and proteins, hereinreferred to as conjugates, that mimic fusion-active transmembraneprotein structures. These conjugates are formed from peptides andproteins that comprise:

-   -   (a) one or more amino acid sequences of 28 or more amino acids        that are capable of forming a stable coiled-coil solution        structure corresponding to or mimicking the heptad repeat region        of gp41; and    -   (b) one or more amino acid sequences that correspond to, or        mimic, an amino acid sequence of the transmembrane-proximal        amphipathic α-helical segment of gp41;        wherein

said one or more sequences (a) and (b) are alternately linked to oneanother via a peptide bond (amide linkage) or by an amino acid linkingsequence consisting of about 2 to about 25 amino acids. These peptidesand proteins are preferably recombinantly produced.

In a preferred embodiment of this aspect of the invention, one or moreof these conjugates folds and assembles into a structure correspondingto, or mimicking, the gp41 core six helix bundle.

Non-limiting examples of the novel constructs or conjugates that can beformed include:

(1) three tandem repeating units consisting of P-17-linker-P-18(P-17-linker-P-18-linker-P-17-linker-P-18-linker-P-17-linker-P-18),

(2) P-17-linker-P-18-linker-P-17,

(3) P-18-linker-P-17-linker-P-18,

(4) P-17-linker-P-17,

(5) three tandem repeating units consisting of P-15-linker-P-16(P-15-linker-P-16-linker-P-15-linker-P-16-linker-P-15-linker-P-16),

(6) P-15-linker-P-16-linker-P-15,

(7) P-16-linker-P-15-linker-P-16, and

(8) P-16-linker-P-15;

wherein each linker is an amino acid sequence, which may be the same ordifferent, of from about 2 to about 25, preferably 2 to about 16 aminoacid residues. Preferred amino acid residues include glycine and serine,for example (GGGGS)_(x), (SEQ ID NO:7) wherein x is 1, 2, 3, 4, or 5, orglycine and cysteine, for example (GGC)y, where y is 1, 2, 3 4 or 5. Inany of the described constructs, P-15 and P-17 are interchangeable andP-16 and P-18 are interchangeable. An example of such a construct (SEQID NO:77) is shown in FIG. 7, along with the corresponding nucleic acidsequence (SEQ ID NO:78) used for recombinant expression of theconstruct.

Alternatively, polyclonal or monoclonal antibodies can be raised againstthe immunogenic mixtures and conjugates described in this aspect of theinvention. Such antibodies can be employed as therapeutic orprophylactic agents.

In preferred aspects of the invention, the methods can be employed toimmunize an HIV-1-infected individual such that levels of HIV-1 will bereduced in such individual. In another aspect, the methods can beemployed to immunize a non-HIV-1-infected individual so that, followinga subsequent exposure to HIV-1 that would normally result in HIV-1infection, the levels of HIV-1 will be non-detectable using currentdiagnostic tests.

Immunogen Preparation

Induction and interpretation of a humoral immune response directedagainst gp41 structural epitopes requires both immunogen preparation andantibody characterization. Synthetic peptides and recombinant proteinscan both be used to generate antigenic structures corresponding to gp41fusion active domains.

In one aspect of the invention, target immunogens model the heptadrepeat region delineated by the P-17 peptide (capable of forming atrimeric coiled-coil structure). In another aspect of the invention,target immunogens model the transmembrane-proximal amphipathic α-helicalsegment delineated by the P-18 peptide. This region in the absence ofthe coiled-coil core exhibits random coil solution structure. (Wild, C.,et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992); Wild, C., etal., AIDS Res. Hum. Retroviruses 9:1051-1053 (1993); Wild, C., et al.,Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994)). In another aspect,combinations of these target immunogens are employed for raisingantibodies.

In another aspect of the invention the target immunogen is the six helixhydrophobic bundle. This bundle is formed by the specific association ofthese two distal regions of the ectodomain of gp41 (Chan, D. C., et al,Cell 89:263-273 (1997); Lu, M., et al., Nature Struct. Biol. 2:1075-1082(1995)). These constructs will mimic entry determinants which form andfunction during HIV-1 entry.

Synthetic Methods of Immunogen Preparation

Immunogens can be prepared by several different routes. The constructscan be generated from synthetic peptides. This involves preparing eachsequence as a peptide monomer followed by post-synthetic modificationsto generate the appropriate oligomeric structures. The peptides aresynthesized by standard solid-phase methodology. To generate a trimericcoiled-coil structure, the P-17 peptide monomer is solubilized underconditions which favor oligomerization. These conditions include a 20 mMphosphate buffer, pH 4.5 and a peptide concentration of 100 μM (Wild,C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541(1992)). Thestructure which forms under these conditions can be optionallystabilized by chemical crosslinking, for example using gluteraldehyde.

Alternatively, a protocol which makes use of intermolecular disulfidebond formation to stabilize the trimeric coiled-coil structure can beemployed in order to avoid any disruptive effect the cross-linkingprocess might have on the structural components of this construct. Thisapproach uses the oxidation of appropriately positioned cysteineresidues within the peptide sequence to stabilize the oligomericstructure. This requires the addition of a short linker sequence to theN terminus of the P-17 peptide. The trimeric coiled-coil structure whichis formed by this approach will be stabilized by the interaction of thecysteine residues (FIG. 2). The trimer is separated from higher orderoligomeric forms, as well as residual monomer, by size exclusionchromatography and characterized by analytical ultracentrifugation.These covalently stabilized coiled-coil oligomers serve as the corestructure for preparation of a six helix bundle.

To accomplish preparation of a six helix bundle, an excess of P-18peptide is added to the purified core structure. After incubation thereaction mixture is subjected to a cross-linking procedure to stabilizethe higher order products of the specific association of these twopeptides. The desired material is isolated by size exclusionchromatography and characterized by analytical ultracentrifugation. Theimmunogen corresponding only to the P-18 peptide requires no specificpost-synthetic modifications. Using this approach, three separate targetconstructs are generated rapidly and in large amounts.

Recombinant Methods of Immunogen Preparation

Another method for preparing target immunogens involves the use of abacterial expression vector to generate recombinant gp41 fragments. Theuse of an expression vector to produce the peptides and polypeptidescapable of forming the entry-relevant immunogens of the presentinvention adds a level of versatility to immunogen preparation.

New and modified forms of the antigenic targets are contemplated as thestructural determinants of HIV-1 entry are better understood. Therecombinant approach readily accommodates these changes. Also, thismethod of preparation allows for the ready modification of the variousconstructs (i.e. the addition of T- or B-cell epitopes to therecombinant gp41 fragments to increase immunogenicity). In addition, aform of the six helix hydrophobic core structure is generated which willnot require additional stabilization, since determining the antigenicnature of this structure is important. Finally, these recombinantconstructs can be employed as a tool to provide valuable insights intoadditional structural components which form and function in gp41 duringthe process of virus entry.

Thus, as part of the invention, novel fusion polypeptides (conjugates)are also provided, as are vectors, host cells and recombinant methodsfor producing the same. The present invention provides isolated nucleicacid molecules comprising a polynucleotide encoding the conjugates ofthe invention.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production offusion polypeptides or peptides by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as that described herein. Other suitable promoters will be known tothe skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

The fusion protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Depending upon the host employedin a recombinant production procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. In addition,polypeptides of the invention may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses.

A bacterial expression vector (kindly provided by Dr. Terrance Oas, DukeUniversity) was developed specifically for the expression of smallproteins. This plasmid, pTCLE-G2C, is based on pAED-4, a T7 expressionvector. A modified TrpLE (Yansura, D. G., Methods Enzymol. 185:161-166(1990)) fusion peptide (provided by Dr. Peter Kim) was inserted afterthe T7 promoter (Studier, F. W., et al., Methods Enzymol. 185:60-89(1990)). There is an in frame Nde I site at the end of the TrpLE peptidethat encodes a methionine cyanogen bromide (CNBr) cleavage site. Thisvector was used in an earlier study to express a recombinant form of theP-17 peptide (Calderone, T. L., et al., J. Mol. Biol. 262:407-412(1996)) and has been modified to expresses the P-18 peptide.

To generate a six helix hydrophobic core structure, several combinationsof the heptad repeat (for example, P-17 or P-15) region and theamphipathic α-helical (for example, P-16 or P-18) segment of gp41 areseparated by a flexible linker of amino acid residues. For example,(GGGGS)₃ (SEQ ID NO:7) can be encoded into the vector. This isaccomplished by standard PCR methods. The (GGGGS)₃ (SEQ ID NO:7) linkermotif is encoded by a synthetic oligonucleotide which is ligated betweenthe P-17 and P-18 encoding regions of the expression vector.

All constructions are characterized by multiple restriction enzymedigests and sequencing. The success of this approach to attainmulticomponent interactions has been recently demonstrated (Huang, B.,et al., J. Immunol. 158:216-225 (1997)).

Examples of the novel constructs or conjugates that can be formed by themethod are described above.

Based on the parallel orientation of the subunits of the coiled coilcore and the antiparallel orientation of the amphipathic α-helicalsegment in the six helix bundle, these constructs fold to generate thedesired structures (See, FIG. 3.). Following expression, the recombinantgp41 fragments are isolated as inclusion bodies, cleaved from the leadersequence by cyanogen bromide, and separated from the leader by-productby size exclusion chromatography step (SUPERDEX 75). This protocol hasbeen successfully used in the purification of large quantities of amodified form of the P-17 peptide (Calderone, T. L., et al., J. Mol.Biol. 262:407-412 (1996)). Recombinant constructs (2) and (3) are mixedin equalmolar quantities under non-denaturing conditions to generate asix-helix hydrophobic core structure. Constructs (1) and (4) will foldeither intra- or intermolecularly to generate the same or similarstructures (see FIG. 3 for the folding process). The desired product ispurified by size exclusion chromatography on a SUPERDEX 75 FPLC columnand characterized by molecular weight under using a Beckman Model XL-Aanalytical ultracentrifuge.

Definitions

The phrase “entry-relevant” as employed herein, refers to particularmolecular conformations or structures that occur or are exposedfollowing interaction of HIV with the cell surface during viral entry,and the role of particular amino acid sequences and molecularconformations or structures in viral entry.

The term “neutralizing” as employed herein refers to the ability toinhibit entry of HIV into cells, including an amount of inhibition thatis useful for reducing or preventing infection of uninfected cells bythe virus.

The term “HIV” as used herein refers to all strains and isolates ofhuman immunodeficiency virus type 1. The constructs of the inventionwere based upon HIV-1 gp41, and the numbering of amino acids in HIVproteins and fragments thereof given herein is with respect to theHIV-1_(LAI) isolate. However, it is to be understood, that while HIV-1viral infection and the effects of the present invention on such HIV-1infection are being used herein as a model system, the entry mechanismthat is being targeted is relevant to all strains and isolates of HIV-1.Hence the invention is directed to “broadly neutralizing” methods.

The phrase “heptad repeat” or “heptad repeat region” as employed herein,refers to a common protein motif having a 4-3 repeat of amino acids,commonly leucine and/or isoleucine, and is often associated withalpha-helical secondary structure. The ‘heptad repeat” can berepresented by the following sequence:

-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-

where AA₁ and AA₄ are each one of leucine or isoleucine; while AA₂, AA₃,AA₅, AA₆, and AA₇ can be any amino acid. See, Wild, C., et al., Proc.Natl. Acad. Sci. USA 89:10537-10541 (1992).

Peptides are defined herein as organic compounds comprising two or moreamino acids covalently joined by peptide bonds. Peptides may be referredto with respect to the number of constituent amino acids, i.e., adipeptide contains two amino acid residues, a tripeptide contains three,etc. Peptides containing ten or fewer amino acids may be referred to asoligopeptides, while those with more than ten amino acid residues arepolypeptides.

Peptides

The complete gp41 amino acid sequence (HIV-1 Group M: Subtype B Isolate:LAI, N to C termini) is:

(SEQ ID NO: 8) AVGIGALFLGFLGAAGSTMGARSMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHISLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLP-TPRG-PDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGL ERILL.

N-Terminal Helix Region:

(SEQ ID NO: 1) ARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKD QQLLGI

Shown below is the sequence for residues 558-595 (SEQ ID NO:7) of theHIV-1_(LAI) gp41 protein in the N-helical domain of the protein. The aand d subscripts denote the 4-3 positions of the heptad repeat.

(SEQ ID NO: 2) N N L L R A I E A Q Q H L L Q L T V W G I K Q L Q    d       a     d       a     d       a     d                           571           578       A R I L A V E R Y L K D Q     a     d       a    585

C-Terminal Helix Region:

(SEQ ID NO: 4) WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN WFNITNW

Shown below is the amino acid sequence for residues 643-678 of theHIV-1_(LAI) gp41 protein in the C-helical domain of the protein.

(SEQ ID NO: 5) Y T S L I H S L I E E S Q N Q Q E K N E Q E L Ld       a    d       a     d       a     d            647          654           661 E L D K W A S L W N W F a     d       a

Unlike the N-helix, when modeled as a peptide, the C-helical region ofgp41 is not structured. However, when mixed with the N-peptide, theC-peptide does takes on α-helical structure as part of the corestructure complex. The structure forms in vitro on mixing the peptidesand can be characterized spectrophotometrically (Lu, M., et al., Nat.Struct. Biol. 2:1075-1082 (1995)). The initial determination of theeffect of the mutations on C-helix structure may be performed byanalyzing the ability of the mutant C-peptide to interact with theN-peptide and form the six-helix bundle. This analysis may be carriedout using circular dichroism. N-helical and C-helical domain peptidescan be constructed from multiple strains of HIV, and can includedeletions, insertions and substitutions that do not destroy the abilityof the resulting peptide to elicit antibodies when employed alone or incombination with other peptides of the invention.

Examples of N-helical Domain Peptide Sequences (All sequences are listedfrom

N-terminus to C-terminus.) from different HIV strains include, but arenot limited to the following peptides:

HIV-1 Group M: Subtype B Isolate: LAI

(SEQ ID NO: 1) ARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKD QQLLGI(SEQ ID NO: 9) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ(SEQ ID NO: 3) P15 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL (SEQ ID NO: 2)P-17  NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ

Subtype B Isolate: ADA

(SEQ ID NO: 10) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYLRDQ(SEQ ID NO: 11) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 12)NNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYLRDQ

Subtype B Isolate: JRFL

(SEQ ID NO: 13) SGIVQQQNNLLRAIEAQQRMLQLTVWGIKQLQARVLAVERYLGDQ(SEQ ID NO: 14) SGIVQQQNNLLRAIEAQQRMLQLTVWGIKQLQRVL (SEQ ID NO: 15)NNLLRAIEAQQRMLQLTVWGIKQLQARVLAVERYLGDQ

Subtype B Isolate: 89.6

(SEQ ID NO: 16) SGIVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARVLALERYLRDQ(SEQ ID NO: 17) SGIVQQQNNLLRAIEAQQHMLQLTVWGIKQLQARVL (SEQ ID NO: 18)NNLLRAIEAQQHMLQLTVWGIKQLQARVLALERYLRDQ

Subtype C Isolate: BU910812

(SEQ ID NO: 19) SGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLRDQ(SEQ ID NO: 20) SGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLARVL (SEQ ID NO: 21)SNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYLRDQ

Subtype D Isolate: 92UG024D

(SEQ ID NO: 22) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVESYLKDQ(SEQ ID NO: 11) SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 23)NNLLRAIEAQQHLLQLTVWIKQLQARVLAVESYLKDQ

Subtype F Isolate: BZ163A

(SEQ ID NO: 24) SGIVQQQSNLLRAIEAQQHLLQLTVWIKQLQARVLAVERYLQDQ(SEQ ID NO: 25) SGIVQQQSNLLRAIEQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 26)SNLLRAIEQQHLLQLTVWGIKQLQARVLAVERYLQDQ

Subtype G Isolate: FI.HH8793

(SEQ ID NO: 27) SGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYDQ(SEQ ID NO: 25) SGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVL (SEQ ID NO: 28)SNLLRAIEAQQHLLQLTVWGIKQLQARVLALERYLRDQ

Subtype H Isolate: BE.VI997

(SEQ ID NO: 29) SGIVQQQSNLLRAIQAQQHMLQLTVWGVKQLQARVLAVERYLKDQ(SEQ ID NO: 30) SGIVQQQSNLLRAIQAQQHMLQLTVWGVKQLQARVL (SEQ ID NO: 31)SNLLRAIQAQQHMLQLTVWGVKQLQARVLAVERYLKDQ

Subtype J Isolate: SE.SE92809

(SEQ ID NO: 32) SGIVQQQSNLLKAIEAQQHLLKLTVWGIKQLQARLAVERYLKDQ(SEQ ID NO: 33) SGIVQQQSNLLKAIEAQQHLLKLTWGIKQLQARVL (SEQ ID NO: 34)SNLLKAIEAQQHLLKLTVWGIKQLQARVLQVERYLKDQ

Group N Isolate: CM.YBF30

(SEQ ID NO: 35) SGIVQQQNILLRAIEAQQHLLQLSIWGIKQLQAKVLAIERYLRDQ(SEQ ID NO: 36) SGIVQQQNILLRAIEAQQHLLQLSIWGIKQLQAKVL  (SEQ ID NO: 37)NILLRAIEAQQHLLQLSIWGIKQLQAKVLAIERYLRDQ

Group O Isolate: CM.ANT70C

(SEQ ID NO: 38) KGIVQQQDNLLRAIQAQQQLLRLSxWGIRQLRARLLALETLLQNQ(SEQ ID NO: 39) KGIVQQQDNLLRAIQAQQQLLRLSxWGIRQLRARL (SEQ ID NO: 40)DNLLRAIQAQQQLLRLSxWGIRQLRARLLALETLLQNQ 

Examples of C-helical Domain Peptide Sequences (All sequences are listedfrom N-terminus to C-terminus.) from different HIV strains include, butare not limited to the following peptides:

HIV-1 Group M: Subtype B Isolate: LAI

(SEQ ID NO: 4) WNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN WFNITNW(SEQ ID NO: 41) WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF(SEQ ID NO: 6) P16 WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL (SEQ ID NO: 5)P-18 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

Subtype B Isolate: ADA

(SEQ ID NO: 42) WMEWEREIENYTGLIYTLIEESQNQQEKNEQDLLALDKWASLWNWF(SEQ ID NO: 43) WMEWEREIENYTGLIYTLIEESQNQQEKNEQDLL (SEQ ID NO: 44)YTGLIYTLIEESQNQQEKNEQDLLALDKWASLWNWF 

Subtype B Isolate: JRFL

(SEQ ID NO: 45) WMEWEREIDNYTSEIYTLIEESQNQQEKNEQELLELDKWASLWNWF(SEQ ID NO: 46) WMEWEREIDNYTSEIYTLIEESQNQQEKNEQELL (SEQ ID NO: 47)YTSEIYTLIEESQNQQEKNEQELLELDKWASLWNWF

Subtype B Isolate: 89.6

(SEQ ID NO: 48) WMEWEREIDNYTDYIYDLLEKSQTQQEKNEKELLELDKWASLWNWF(SEQ ID NO: 49) WMEWEREIDNYTDYIYDLLEKSQTQQEKNEKELL (SEQ ID NO: 50)YTDYIYDLLEKSQTQQEKNEKELLELDKWASLWNWF

Subtype C Isolate: BU910812

(SEQ ID NO: 51) WIQWDREISNYTGIIYRLLEESQNQQENNEKDLLALDKWQNLWSWF(SEQ ID NO: 52) WIQWDREISNYTGIIYRLLEESQNQQENNEKDLL (SEQ ID NO: 53)YTGIIYRLLEESQNQQENNEKDLLALDKWQNLWSWF

Subtype D Isolate: 92UG024D

(SEQ ID NO: 54) WMEWEREISNYTGLIYDLIEESQIQQEKNEKDLLELDKWASLWNWF(SEQ ID NO: 55) WMEWEREISNYTGLIYDLIEESQIQQEKNEKDLL (SEQ ID NO: 56)YTGLIYDLIEESQIQQEKNEKDLLELDKWASLWNWF

Subtype F Isolate: BZ163A

(SEQ ID NO: 57) WMEWQKEISNYSNEVYRLIEKSQNQQEKNEQGLLALDKWASLWNWF(SEQ ID NO: 58) WMEWQKEISNYSNEVYRLIEKSQNQQEKNEQGLL (SEQ ID NO: 59)YSNEVYRL1EKSQNQQEKNEQGLLALDKWASLWNWF

Subtype G Isolate: FI.HH8793

(SEQ ID NO: 60) WIQWDREISNYTQQIYSLIEESQNQQEKNEQDLLALDNWASLWTWF(SEQ ID NO: 61) WIQWDREISNYTQQIYSLIEESQNQQEKNEQDLL (SEQ ID NO: 62)YTQQIYSLIEESQNQQEKNEQDLLALDNWASLWTWF

Subtype H Isolate: BE.VI997

(SEQ ID NO: 63) WMEWDRQIDNYTEVIYRLLELSQTQQEQNEQDLLALDKWDSLWNWF(SEQ ID NO: 64) WMEWDRQIDNYTEVIYRLLELSQTQQEQNEQDLL (SEQ ID NO: 65)YTEVIYRLLELSQTQQEQNEQDLLALDKWDSLWNWF

Subtype J Isolate: SE.SE92809

(SEQ ID NO: 66) WIQWEREINNYTGIIYSLLEEAQNQQENNEKDLLALDKWTNLWNWFN(SEQ ID NO: 67) WIQWEREINNYTGIIYSLIEEAQNQQENNEKDLL (SEQ ID NO: 68)YTGIIYSLIEEAQNQQENNEKDLLALDKWTNLWNWFN

Group N Isolate: CM.YBF30

(SEQ ID NO: 69) WQQWDEKVRNYSGVIFGLIEQAQEQQNTNEKSLLELDQWDSLWSWF(SEQ ID NO: 70) WQQWDEKVRNYSGVIFGLIEQAQEQQNTNEKSLL (SEQ ID NO: 71)YSGVIFGLIEQAQEQQNTNEKSLLELDQWDSLWSWF

Group O Isolate: CM.ANT70C

(SEQ ID NO: 72) WQEWDRQISNISSTIYEEIQKAQVQQEQNEKKLLELDEWASIWNWL(SEQ ID NO: 73) WQEWDRQISNISSTIYEEIQKAQVQQEQNEKKLL (SEQ ID NO: 74)ISSTIYEEIQKAQVQQEQNEKKLLELDEWASIWNWL

The peptides and conjugates of the present invention may be acylated atthe NH₂ terminus, and may be amidated at the COOH terminus.

The peptides and conjugates of the invention may include conservativeamino acid substitutions. Conserved amino acid substitutions consist ofreplacing one or more amino acids of the peptide sequence with aminoacids of similar charge, size, and/or hydrophobicity characteristics,such as, for example, a glutamic acid (E) to aspartic acid (D) aminoacid substitution. When only conserved substitutions are made, theresulting peptide is functionally equivalent to the peptide from whichit is derived.

Peptide sequences defined herein are represented by one-letter symbolsfor amino acid residues as follows:

-   A alanine-   R arginine-   N asparagine-   D aspartic acid-   C cysteine-   Q glutamine-   E glutamic acid-   G glycine-   H histidine-   I isoleucine-   L leucine-   K lysine-   M methionine-   F phenylalanine-   P proline-   S serine-   T threonine-   W tryptophan-   Y tyrosine-   V valine

The peptides and conjugates of the invention may include amino acidinsertions which consist of single amino acid residues or stretches ofresidues ranging from 2 to 15 amino acids in length. One or moreinsertions may be introduced into the peptide, peptide fragment, analogand/or homolog.

The peptides and conjugates of the invention may include amino aciddeletions of the full length peptide, analog, and/or homolog. Suchdeletions consist of the removal of one or more amino acids from thefull-length peptide sequence, with the lower limit length of theresulting peptide sequence being 4 to 6 amino acids. Such deletions mayinvolve a single contiguous portion or greater than one discrete portionof the peptide sequences.

The peptides of the invention may be synthesized or prepared bytechniques well known in the art. See, for example, Creighton, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., New York, N.Y.(1983), which is incorporated herein by reference in its entirety. Shortpeptides, for example, can be synthesized as a solid support or insolution. Longer peptides maybe made using recombinant DNA techniques.Here, the nucleotide sequences encoding the peptides of the inventionmay be synthesized, and/or cloned, and expressed according to techniqueswell known to those of ordinary skill in the art. See, for example,Sambrook, et al., Molecular Cloning, A Laboratory Manual, Vols. 1-3,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

In yet another embodiment of the invention, peptides comprising thesequences described above may be synthesized with additional chemicalgroups present at their amino and/or carboxy termini, such that, forexample, the stability, bioavailability, and/or immunogenic activity ofthe peptides is enhanced. For example, hydrophobic groups such ascarbobenzoxy, dansyl, or t-butyloxycarbonyl groups, may be added to thepeptides' amino termini. Likewise, an acetyl group or a9-fluorenylmethoxy-carbonyl group may be placed at the peptides' aminotermini. Additionally, the hydrophobic group t-butyloxycarbonyl, or anamido group may be added to the peptides' carboxy termini. In onepreferred embodiment, carrier proteins, such as keyhole limpethemocyanin, ovalbumin, BSA or tetanus toxoid are added to the peptide.

With reference to the peptides P-17 and P-18, deletion mutants arefurther described.

The peptide P-18 corresponds to amino acid residues 638 to 673 of thetransmembrane protein gp41 from the HIV-1_(LAI) isolate:

In addition to the full-length C-helical peptides identified above,useful peptides of the invention may include truncations of theC-helical peptides (SEQ ID NO:4) which exhibit the ability to raiseneutralizing antibodies or form a six-helix hydrophobic core structureunder conditions described herein. Such truncated peptides may comprisepeptides of between 3 and 56 amino acid residues, i.e., peptides rangingin size from a tripeptide to a 56-mer polypeptide. As an example, suchpeptides are listed for P-18 in Tables I and II, below. Peptidesequences in these tables are listed from amino (left) to carboxy(right) terminus. “X” may represent an amino group (—NH₂) and “Z” mayrepresent a carboxyl (—COOH) group. Alternatively, as described below,“X” and/or “Z” may represent a hydrophobic group, an acetyl group, aFMOC group, an amido group, or a covalently attached macromolecule.

TABLE I Carboxy Truncations of SEQ ID NO: 5 X-YTS-Z  X-YTSL-Z X-YTSLI-ZX-YTSLIH-Z X-YTSLIHS-Z X-YTSLIHSL-Z X-YTSLIHSLI-Z X-YTSLIHSLIE-ZX-YTSLIHSLIEE-Z X-YTSLIHSLIEES-Z X-YTSLIHSLIEESQ-Z X-YTSLIHSLIEESQN-ZX-YTSLIHSLIEESQNQ-Z X-YTSLIHSLIEESQNQQ-Z X-YTSLIHSLIEESQNQQE-ZX-YTSLIHSLIEESQNQQEK-Z X-YTSLIHSLIEESQNQQEKN-Z X-YTSLIHSLIEESQNQQEKNE-ZX-YTSLIHSLIEESQNQQEKNEQ-Z X-YTSLIHSLIEESQNQQEKNEQE-ZX-YTSLIHSLIEESQNQQEKNEQEL-Z X-YTSLIHSLIEESQNQQEKNEQELL-ZX-YTSLIHSLIEESQNQQEKNEQELLE-Z X-YTSLIHSLIEESQNQQEKNEQELLEL-ZX-YTSLIHSLIEESQNQQEKNEQELLELD-Z X-YTSLIHSLIEESQNQQEKNEQELLELDK-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKW-Z X-YTSLIHSLIEESQNQQEKNEQELLELDKWA-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWAS-Z X-YTSLIHSLIEESQNQQEKNEQELLELDKWASL-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLW-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWN-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Z The one letter amino acid codeis used. “X” may represent a hydrogen attached to the terminal aminogroup, an amino protecting group including, but not limited to,carbobenzoxyl, dansyl, or t-butyloxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier groupincluding, but not limited to, lipid-fatty acid conjugates, polyethyleneglycol, or carbohydrates. “Z” may represent a terminal carboxyl (COOH);an amido group; an ester group (COOR) including, but not limited to, at-butyloxycarbonyl group; a macromolecular carrier group including, butnot limited to, lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates.

TABLE II Amino Truncations of SEQ ID NO: 5 X-NWF-Z X-WNWF-Z X-LWNWF-ZX-SLWNWF-Z X-ASLWNWF-Z X-WASLWNWF-Z X-KWASLWNWF-Z X-DKWASLWNWF-ZX-LDKWASLWNWF-Z X-ELDKWASLWNWF-Z X-LELDKWASLWNWF-Z X-LLELDKWASLWNWF-ZX-ELLELDKWASLWNWF-Z X-QELLELDKWASLWNWF-Z X-EQELLELDKWASLWNWF-ZX-NEQELLELDKWASLWNWF-Z X-KNEQELLELDKWASLWNWF-Z X-EKNEQELLELDKWASLWNWF-ZX-QEKNEQELLELDKWASLWNWF-Z X-QQEKNEQELLELDKWASLWNWF-ZX-NQQEKNEQELLELDKWASLWNWF-Z X-QNQQEKNEQELLELDKWASLWNWF-ZX-SQNQQEKNEQELLELDKWASLWNWF-Z X-ESQNQOEKNEQELLELDKWASLWNWF-ZX-EESQNQQEKNEQELLELDKWASLWNWF-Z X-IEESQNQQEKNEQELLELDKWASLWNWF-ZX-LIEESQNQQEKNEQELLELDKWASLWNWF-Z X-SLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-HSLIEESQNQQEKNEQELLELDKWASLWNWF-Z X-IHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-LIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-SLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-ZX-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Z The one letter amino acid codeis used. “X” may represent a hydrogen attached to the terminal aminogroup, an amino protecting group including, but not limited to,carbobenzoxyl, dansyl, or t-butyloxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier groupincluding, but not limited to, lipid-fatty acid conjugates, polyethyleneglycol, or carbohydrates. “Z” may represent a terminal carboxyl (COOH);an amido group; an ester group (COOR) including, but not limited to, at-butyloxycarbonyl group; a macromolecular carrier group including, butnot limited to, lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates.

The peptides also include analogs of which may include, but are notlimited to, peptides comprising the a full-length or truncated sequence,containing one or more amino acid substitutions, insertions and/ordeletions.

There exists a striking amino acid conservation within the C-helicalregions of HIV-1 and HIV-2. The amino acid conservation is of a periodicnature, suggesting some conservation of structure and/or function. Auseful peptide derived from the HIV-2_(NHZ) isolate has the 36 aminoacid sequence (reading from amino to carboxy terminus):

(SEQ ID NO: 5) NH₂-LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL-COOH

Further, peptides useful for forming “entry-relevant” structures includepeptides corresponding to the N-helical domain of gp41. One example ofsuch a peptide, P-17, corresponds to residues 558 to 595 of thetransmembrane protein gp41 from the HIV-1_(LAI) isolate.

In addition to the full-length N-helical peptides (for example, (SEQ IDNO:1)) shown above, the peptides may include truncations of thesepeptides which exhibit the ability to form stable coiled-coil structure.Such truncated peptides may comprise peptides of between 3 and 55 aminoacid residues, i.e., peptides ranging in size from a tripeptide to a55-mer polypeptide, as shown in Tables III and IV, below for P-17.Peptide sequences in these tables are listed from amino (left) tocarboxy (right) terminus. “X” may represent an amino group (—NH₂) and“Z” may represent a carboxyl (—COOH) group. Alternatively, “X” and/or“Z” may represent a hydrophobic group, an acetyl group, a FMOC group, anamido group or a covalently attached macromolecular group.

TABLE III Carboxy Truncations of SEQ ID NO: 2 X-NNL-Z X-NNLL-Z X-NNLLR-ZX-NNLLRA-Z X-NNLLRAI-Z X-NNLLRAIE-Z X-NNLLRAIEA-Z X-NNLLRAIEAQ-ZX-NNLLRAIEAQQ-Z X-NNLLRAIEAQQH-Z X-NNLLRAIEAQQHL-Z X-NNLLRAIEAQQHLL-ZX-NNLLRAIEAQQHLLQ-Z X-NNLLRAIEAQQHLLQL-Z X-NNLLRAIEAQQHLLQLT-ZX-NNLLRAIEAQQHLLQLTV-Z X-NNLLRAIEAQQHLLQLTVW-Z X-NNLLRAIEAQQHLLQLTVWQ-ZX-NNLLRAIEAQQHLLQLTVWQI-Z X-NNLLRAIEAQQHLLQLTVWQIK-ZX-NNLLRAIEAQQHLLQLTVWQIKQ-Z X-NNLLRAIEAQQHLLQLTVWQIKQL-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQ-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQA-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQAR-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQARI-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARIL-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQARILA-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAV-Z X-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVE-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVER-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERY-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYL-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLK-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKD-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z The one letter amino acidcode is used. “X” may represent a hydrogen attached to the terminalamino group, an amino protecting group including, but not limited to,carbobenzoxyl, dansyl, or t-butyloxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier groupincluding, but not limited to, lipid-fatty acid conjugates, polyethyleneglycol, or carbohydrates. “Z” may represent a terminal carboxyl (COOH);an amido group; an ester group (COOR) including, but not limited to, at-butyloxycarbonyl group; a macromolecular carrier group including, butnot limited to, lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates.

TABLE IV Amino Truncations of SEQ ID NO: 2                                   X-KDQ-Z                                  X-LKDQ-Z                                 X-YLKDQ-Z                                X-RYLKDQ-Z                               X-ERYLKDQ-Z                              X-VERYLKDQ-Z                             X-AVERYLKDQ-Z                            X-LAVERYLKDQ-Z                           X-ILAVERYLKDQ-Z                          X-RILAVERYLKDQ-Z                         X-ARILAVERYLKDQ-Z                        X-QARILAVERYLKDQ-Z                       X-LQARILAVERYLKDQ-Z                      X-QLQARILAVERYLKDQ-Z                     X-KQLQARILAVERYLKDQ-Z                    X-IKQLQARILAVERYLKDQ-Z                   X-QIKQLQARILAVERYLKDQ-Z                  X-WQIKQLQARILAVERYLKDQ-Z                 X-VWQIKQLQARILAVERYLKDQ-Z                X-TVWQIKQLQARILAVERYLKDQ-Z               X-LTVWQIKQLQARILAVERYLKDQ-Z              X-QLTVWQIKQLQARILAVERYLKDQ-Z             X-LQLTVWQIKQLQARILAVERYLKDQ-Z            X-LLQLTVWQIKQLQARILAVERYLKDQ-Z           X-HLLQLTVWQIKQLQARILAVERYLKDQ-Z          X-QHLLQLTVWQIKQLQARILAVERYLKDQ-Z         X-QQHLLQLTVWQIKQLQARILAVERYLKDQ-Z        X-AQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z       X-EAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z      X-IEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z     X-AIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z    X-RAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z   X-LRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z  X-LLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z X-NLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-ZX-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-Z The one letter amino acidcode is used. “X” may represent a hydrogen attached to the terminalamino group, an amino protecting group including, but not limited to,carbobenzoxyl, dansyl, or t-butyloxycarbonyl; an acetyl group; a9-fluorenylmethoxy-carbonyl (FMOC) group; a macromolecular carrier groupincluding, but not limited to, lipid-fatty acid conjugates, polyethyleneglycol, or carbohydrates. “Z” may represent a terminal carboxyl (COOH);an amido group; an ester group (COOR) including, but not limited to, at-butyloxycarbonyl group; a macromolecular carrier group including, butnot limited to, lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates.

The N-helical peptides also include analogs and/or truncations which mayinclude, but are not limited to, peptides comprising the full-length ora truncated sequence, containing one or more amino acid substitutions,insertions and/or deletions.

Antibody Generation and Characterization

Generation and characterization of the antibodies generated againstnovel gp41 epitopes constitutes the second aspect of the invention. Theexperimental sera and monoclonal antibodies generated against the targetimmunogens are subjected to thorough biophysical and biologicalevaluation.

Antibodies are generated following established protocols. All smallanimal work (immunizations, bleeds, and hybridoma production) is carriedout by standard methods known to those of skill in the art. A first setof immunogens consists of the peptide constructs P-15 or P47 (capable offorming trimeric coiled-coil multimers, optionally stabilized bychemical cross-linking or oxidation), P-16 or P48, and the P-17/P-18mixture or P-15/P-16 mixture (wherein the peptides are optionallychemically or oxidatively cross-linked). In one set of experiments, theimmunogens are conjugated to a carrier such as KLH.

Balb-c mice are immunized with each of these constructs. Due to possibledisruptive effects of carrier conjugation on antigen structure, onegroup of mice from each set can be immunized with 100 μg of unconjugatedpeptide, while another group of mice can receive 100 μg of antigenconjugated to KLH. Following the initial immunization the animalsreceive a 100 μg boost on day 14 followed by 50 μg boosts on days 30 and45. Bleeds occur two weeks following the final boost. Mice are alsoimmunized with the recombinant constructs following the same outline asthat for the peptide immunogens.

Alternative immunization approaches include the use of a recombinantadenovirus vector expressing all or part of the HIV-1 envelopeglycoprotein gp120/gp41 as the primary immunogen followed by boosterimmunizations with the gp41 peptides, proteins or other constructs.

The polyclonal sera generated by the immunization of experimentalanimals undergo an initial screen for virus inhibition. Antiviralactivity is evaluated in both cell-cell fusion and neutralizationassays. In this second format, a representative sample of lab adaptedand primary virus isolates is used. Both assays are carried outaccording to protocols described previously (Wild, C., et al., Proc.Natl. Acad Sci. USA 89:10537-10541 (1992); Wild, C., et al., Proc. Natl.Acad. Sci. USA 91:12676-12680 (1994); Wild, C., et al., Proc. Natl.Acad. Sci. USA 91:9770-9774 (1994)). Samples are also screened by ELISAto characterize binding. The antigen panel includes all experimentalimmunogens. Animals with sera samples which test positive for binding toone or more experimental immunogens are candidates for use in MAbproduction. Following this initial screen, one animal representing eachexperimental immunogen is selected for monoclonal antibody production.The criteria for this selection is based on neutralizing antibody titersand in the absence of neutralization, binding patterns against the panelof structured immunogens.

Hybridoma supernatants are screened by ELISA, against structured andnon-structured peptides and recombinants. Samples that are ELISAnegative or weakly positive are further characterized for IgG. If IgG ispresent the material is screened in the biophysical and biologicalassays. Strongly positive samples are screened for their ability toneutralize virus and bind envelope. The experimental material can befurther tested against a panel representing the spectrum of HIV-1isolates. These isolates include lab adapted and primary virus strains,syncytium and non-syncytium inducing isolates, virus representingvarious geographic subtypes, and viral isolates which make use of therange of second receptors during virus entry. These neutralizationassays employ either primary cell and cell line targets as required.

Antibodies are characterized in detail for their ability to bind HIVenvelope under various conditions. It is another object of the inventionto determine the gp41 target epitopes are exposed on native envelope orif the envelope must first undergo some interaction which triggers aconformational change i.e binding CD4 and/or co-receptor in order toexpose these epitopes. For detection of antibody binding to nativeenvelope, immunoprecipitations on Env-expressing cells and virions, bothintact and lysed are performed using non-ionic detergents (Furata, R Aet al., Nat. Struct. Biol. 5(4):276-279 (1997); White, J. M. and I. A.Wilson, J. Cell Biol. 105:2887-2894 (1987); Kemble, G. W., et al., J.Virol. 66:4940-4950 (1992)). Antibody binding to cell lysates and intactvirions are also assayed in an ELISA format. Flow cytometry experimentsare performed to determine binding to envelope expressing cells.Cross-competition experiments using other mapped Mabs, human sera, andpeptides can also be performed. To characterize “triggers” to theconformational change, antibody binding to virus in the presence andabsence of both sCD4 and target cells can be compared (White, J. M. andI. A. Wilson, J. Cell Biol. 105:2887-2894 (1987); Kemble, G. W., et al.,J. Virol. 66:4940-4950 (1992)). Because the gp41 regions are highlyconserved, epitope exposure using several different envelopes can becompared to discern possible differences in structure between primary,lab-adapted and genetically diverse virus isolates.

Pharmaceutical Compositions and Methods of Using

The immunogenic constructs of the present invention can be employed invaccines in an amount effective depending on the route ofadministration. Although subcutaneous or intramuscular routes ofadministration are preferred, peptides, multimers or peptide conjugatesof the present invention can also be administered by an intraperitonealor intravenous route. One skilled in the art will appreciate that theamounts to be administered for any particular treatment protocol can bereadily determined without undue experimentation.

The vaccines of the present invention may be employed in such forms ascapsules, liquid solutions, suspensions or elixirs for oraladministration, or sterile liquid forms such as solutions orsuspensions. Any inert carrier is preferably used, such as saline,phosphate-buffered saline, or any such carrier in which the conjugatevaccine has suitable solubility properties. The vaccines may be in theform of single dose preparations or in multi-dose flasks which can beused for mass vaccination programs. Reference is made to Remington'sPharmaceutical Sciences, Osol, ed., Mack Publishing Co., Easton, Pa.(1980), and New Trends and Developments in Vaccines, Voller, et al.,eds., University Park Press, Baltimore, Md. (1978), for methods ofpreparing and using vaccines.

The vaccines of the present invention may further comprise adjuvantswhich enhance production of HIV-specific antibodies. Such adjuvantsinclude, but are not limited to, various oil formulations such asFreund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Pat.No. 4,258,029), the dipeptide known as MDP, saponins and saponinderivatives, such as Quil A and QS-21, aluminum hydroxide, and lymphaticcytokine. Preferably, an adjuvant will aid in maintaining the secondaryand quaternary structure of the immunogens.

Freund's adjuvant is an emulsion of mineral oil and water which is mixedwith the immunogenic substance. Although Freund's adjuvant is powerful,it is usually not administered to humans. Instead, the adjuvant alum(aluminum hydroxide) or ST may be used for administration to a human.The vaccine may be absorbed onto the aluminum hydroxide from which it isslowly released after injection. The vaccine may also be encapsulatedwithin liposomes according to Fullerton, U.S. Pat. No. 4,235,877, ormixed with or liposomes or lipid mixtures to provide an environmentsimilar to the cell surface environment.

In another preferred embodiment, one or more immunogens of the inventionare combined with other immunogens that are used to vaccinate animals.

In another preferred embodiment, the present invention relates to amethod of inducing an immune response in an animal comprisingadministering to the animal the vaccine of the invention in an amounteffective to induce an immune response. Optionally, the vaccine of theinvention may be coadministered with effective amounts of otherimmunogens as mentioned above to generate multiple immune responses inthe animal.

Compositions of the invention are useful as vaccines to induce activeimmunity towards antigens in subjects. Any animal that may experiencethe beneficial effects of the compositions of the present inventionwithin the scope of subjects that may be treated. The subjects arepreferably mammals, and more preferably humans.

The administration of the vaccine may be for either a “prophylactic” or“therapeutic” purpose. When provided prophylactically, the vaccine(s)are provided in advance of any symptoms of HIV infection, or in advanceof any known exposure to HIV. The prophylactic administration of thevaccine(s) serves to prevent or attenuate any subsequent infection. Whenprovided therapeutically, the vaccine(s) is provided upon or after thedetection of symptoms which indicate that an animal may be infected withHIV, or upon or after exposure to the virus. The therapeuticadministration of the vaccine(s) serves to attenuate any actualinfection, for example as measured by improving the symptoms of asubject, or by reducing the level of viral replication. Thus, thevaccines, may be provided either prior to the onset of infection (so asto prevent or attenuate an anticipated infection) or after theinitiation of an actual infection.

EXAMPLE 1 Immunogens of the Present Invention Elicit a NeutralizingAntibody Response to Entry-Relevant Structures on HIV-I gp41

Materials and Methods

Polyclonal sera can be obtained by immunizing rabbits or guinea pigsusing methods well known to those skilled in the art. For example, theanimals are immunized at multiple sites (sub-cutaneous andsub-clavicular) with a total of 200 μg (rabbits) or 100 μg (guinea pigs)of the appropriate peptide, protein, combination of construct incomplete Freund's adjuvant. This is followed by two boosterimmunizations with the same immunogen in incomplete Freund's adjuvant atmonthly intervals following the primary immunization. Sera are collectedprior to, and at intervals following, the series of immunizations. Thesesera are analyzed for the presence of antibodies to the immunogen orother antigens by various assays including those described below.

-   Peptide ELISA: Antigen was coated onto 96-well microtiter plates    (Immulon 2) at 1 μg/well. Following overnight incubation at 4° C.,    plates were washed, blocked and test sera was added. After a 1.5 hr    incubation, plates are washed and bound antibody is detected by    addition of phosphatase-conjugated secondary antibody and    development by pNPP.-   Dot Blots: Antigen (2 μg) was blotted onto nitrocellulose, blocked    and allowed to air dry. Blots were incubated 4 hr with test sera at    a 1:100 dilution. A secondary antibody peroxidase/TMB detection    system was used.-   Western Blots: Immunoblots were carried out using commercially    available strips (Organon Teknika) with test sera at 1:100.-   Viral Lysate Immunoprecipitation: HIV-1 infected cells (IIIB/H9)    were lysed and mixed with immune sera at a dilution of 1:100.    Following incubation with Protein A-agarose, immunoprecipitates were    separated by SDS-PAGE and probed with a gp41 specific monoclonal    antibody.-   Cell Surface Immunoprecipitation: Two days post transfection,    1.5×10⁷ envelope expressing 293T cells were incubated with    experimental sera with and without sCD4 (10 μg/ml unless otherwise    noted). Following incubation, cells were lysed and incubated with    Protein A-agarose. Immunoprecipitates were separated by SDS-PAGE and    probed with a gp41 specific monoclonal antibody.-   Neutralization Assay: Test sera was incubated at a 1:10 dilution    with indicated amount of virus (HIV-1 IIIB) for 1 hr at 37° C. At    the end of this time target cells were added (CEM) and the    experiment was returned to the incubator. On days 1, 3 and 5,    post-infection complete media changes were carried out. On day 7 PI    culture supernatant were harvested. Levels of virus replication were    determined by p24 antigen capture. Levels of replication in test    wells were normalized to virus only controls.

Results

Rabbits or guinea pigs were immunized and sera analyzed by methodsdescribed above. The following data describe the characterization ofpolyclonal antibodies generated to various immunogens that are thesubject of this invention.

Table V illustrates results of the analysis of polyclonal sera tovarious immunogens analyzed by peptide ELISA or dot blots. Severalimmunogens elicited a strong antibody response in these assays. Forexample, immunization with P15 resulted in sera with strong antibodyreactivity to P15 by peptide ELISA (titer>1:102400), and strongreactivity to P15, a mixture of P15+P16 and HIV-1 gp41 by dot blot.Similar results were obtained in these assays following immunizationwith a mixture of P15 and P16 (Table V).

Description of Table V: Analysis of polyclonal sera to variousimmunogens by peptide ELISA or dot blot. For this and subsequent figuresall results are based on immunizations of rabbits except forimmunizations with P-17 or P-18 alone which were performed in guineapigs. The immunogens used are indicated in the vertical list on the leftside of the table. The antigens used in each assay are indicated on thetop row of the table. Peptide ELISA results are presented as titers (themaximum dilution that gives a positive result in the assay). Dot blotresults are scored from—(no reactivity) to +++ (very strong reactivity).HIV TM is HIV-1 gp41. For Table V, *BS³ refers to chemically crosslinked; and ND indicates “not determined.”

TABLE VA Dot Blot Immunogen P15 P16 P15 + P16 P-17 P-18 HIV TM P15 +++ −+++ + − +++ P-17 ND ND ND ND ND ND P16 − +/− ++ − − + P-18 ND ND ND NDND ND P15 + P16 +++ + +++ +/− +/− +++ P-17 + P-18 − − − ++ +/− + P-17 +P-18* − − − +++ +/− ++ P15* +++ − +++ + − ++ P16* − + ++ +/− − ++ HIV TMND ND ND ND ND ND

TABLE Vb Peptide ELISA Immunogen P15 P16 P15 + P16 P-17 P-18 P-17/P-18P-15/P-18 gp 41 P15 1:1.6 × 10⁶ 1:1600     1:1.6 × 10⁶ ~1:800 ND ND ND1:6400 P-17 ND ND ND     1:4.1 × 10⁵ ND 1:4.1 × 10⁵ ND  1:25600 P16>1:1600    1:1.0 × 10⁵ 1:25600    ND ~1:800  ND ND 1:1600 P-18 ND ND NDND  1:25600 1:1.0 × 10⁵ ND  1:25,600 P15 + P16 1:4.1 × 10⁵ 1:4.1 × 10⁵1:4.1 × 10⁵ <1:100 >1:3200  ND ND     1:4.1 × 10⁵ P-17 + P-18 ND ND>1:200        1:25600 1:6400 1:1.0 × 10⁵ ND 1:1600 P-17 + P-18* ND ND>1:100      1:1600 <1:1600  ND ND ND P15* 1:12800    ND >1:25600    NDND ND ND ND P16* ND 1:25600    >1:25600    ND ND ND ND ND P-15/P-181:4.1 × 10⁵ ND ND ND 1:6400 ND 1:4.1 × 10⁵  1:25600 HIV TM 1:25600   >1:6400    ND  1:400 1:1600

These results were confirmed and extended by analysis of the polyclonalsera for reactivity with HIV-1 gp120, gp41 or gp160 by western blot orimmunoprecipitation (Table VI). For example, immunization with P15 orP15+P16 elicited antibodies that reacted with gp160 by western blot, andwhich precipitated gp41 in infected cell lysates. Of particularinterest, P15+P16 elicited an immune response that reacted with cellsurface gp41, but only following treatment of the cells with sCD4 (FIG.4). Previous reports have found that sCD4 binds to gp120 resulting inconformational changes in gp120/gp41 or stripping of gp120 from gp41.This process presumably mimics events that occur at attachment of HIV-1to its receptor CD4 on target cells. The present results suggest thatimmunization with the mixture of P15+P16 elicits an immune response tocryptic epitopes on gp41 that are only exposed following binding ofgp120 to CD4. Table VI: Analysis of polyclonal sera to variousimmunogens by western blot or immunoprecipitation. The immunogens usedare indicated in the vertical list on the left side of the table. Theantigens used in each assay are indicated on the top row of the table.Results are scored from—(no reactivity) to ++++ (very strongreactivity), w: weak reactivity; *: BS³ chemically cross-linked prior toadministration; ND: not determined; HIV TM: HIV-1 gp41.

TABLE VI HIV-1 WB IP gp160 gp41 Lysate Surface P15 w w ++++ − P-17 w w++++ ND P16 − − − − P-18 + w − ND P15 + P16 +++ ++ ++++ + P-17 + P-18 −++ − ND P-17 + P-18* w − − ND P15* w − +++ − P16* + − − ND HIV TM +++ ++++++ ND

FIG. 5 provides data demonstrating that immunogens of the presentinvention elicit a neutralizing antibody response. While somenon-specific inhibition of HIV-1 replication is seen followingincubation with pre-bleed sera, considerably greater inhibition is seenfollowing incubation with sera from animals immunized with P15 orP15+P16. These results indicate that these sera contain neutralizingantibodies resulting from immunization with the immunogen of, and by themethods of, the current invention.

These data are supported by the fact that monoclonal antibodies havebeen generated in mice to several of the immunogens discussed above.When analyzed by some of the methods described above similar resultswere obtained to those seen with the polyclonal sera (not shown).

Discussion

The structural components of gp41, which are present only during virusentry, form a novel set of neutralizing epitopes. The relatively shortlived nature of these entry relevant structures and their presence onlyduring natural infection would account for the observation thatneutralizing antibodies targeting gp41 epitopes are poorly representedin sera from HIV infected individuals and all but absent in vaccineesera. This theory is supported by work involving synthetic peptideswhich model the regions of gp41 identified as taking part in the entryrelated structural reorganization (Wild, C., et al., Proc. Natl. Acad.Sci. USA 89:10537-10541 (1992); Wild, C., et al., AIDS Res. Hum.Retroviruses 9:1051-1053 (1993); Wild, C., et al., Proc. Natl. Acad.Sci. USA 91:12676-12680 (1994); Wild, C., et al., AIDS Res. Hum.Retroviruses 11:323-325 (1995); Wild, C., et al., Proc. Natl. Acad. Sci.USA 91:9770-9774 (1994)). It has been shown that these materials inhibitHIV infection by blocking virus entry and the mechanism of theiractivity is their ability to interact with and disrupt gp41 structuralcomponents critical to the entry event. Although transitory, these gp41entry structures are both accessible and appropriately sensitive targetsfor neutralizing antibody.

Independent of their neutralizing potential, monoclonal antibodiestargeted to conserved structures in the TM will prove invaluable asreagents for dissecting the structural transitions that occur in Env aspart of virus entry.

We have been successful in our initial attempt to generate a structurespecific antibody against the coiled-coil region of gp41. In this workwe used a modified form of the P-17 peptide as immunogen and generatedMAbs that recognize the structured peptide but not a proline containingP-17 analog which is unstructured. Also, this antibody canco-immunoprecipitate the P-18 peptide.

EXAMPLE 2 Neutralizing Antibody Response to Peptides Modeling theC-Helical Region of gp41

This example measures the humoral response to antigens modeling theC-region of gp41. This work used synthetic peptides and a recombinantform of viral protein to characterize antibodies raised against theC-helical regions of gp41 of the viral TM.

These studies employ antibody binding assays to determine the ability ofthese materials to generate an immune response to various forms ofenvelope (native vs. denatured) and virus neutralization assays tocharacterize the antibody response raised against these gp41 domains.The complete panel of immunogens has generated data which allow newinsight into the antigenic nature of gp41. Most encouraging have beenthe results from Guinea Pigs immunized with the peptide, P-18, modelingthe C-helix entry domain (amino acid residues 643-678 of gp41).Specifically, two of three animals receiving this material exhibited aneutralizing antibody response against divergent virus isolates in avariety of assay formats. Additional studies have confirmed theseresults. See Example 3.

In study 1, guinea pigs were immunized intramuscularly with 100 μg ofP-18 formulated in either Freund's complete (prime) or incomplete(boost) adjuvant. Animals were immunized on days 0, 21, 34, 48 and 62.Blood was collected on days 44, 58 and 72. In the initial screen, seraat at 1:10 dilution were tested for ability to inhibit virus-inducedcell killing. In these assays two of the three animals receiving theP-18 peptide (guinea pigs gp233 and gp234) were able to block thecytopathic effects of a pair of prototype HIV-1 isolates. Against the MNisolate >80% protection was achieved while against RF protection was>50%.

In an assay employing the same format (against HIV-1_(MN)), we titratedthe sera from gp233 and gp234. As can be seen in FIG. 6 a, these animalsdisplayed the expected dose-related anti-viral activity. Guinea pigs 233and 234 gave a 50% reduction in virus-induced cell killing at 1:40 and1:37 dilutions, respectively.

A neutralization assay was carried out employing a different target celland endpoint analysis. In this format, CEM T-cell line was inoculatedwith 200 TCID₅₀ of the HIV-1_(MN) isolate. The reduction in viralreplication for gp233 and gp 234 at a serum dilution of 1:10 is shown inFIG. 6 b.

FIG. 6 a shows the titration of bleed 2 for each animal againstHIV-1_(MN) in the cell killing assay which uses cell viability as ameasure of virus neutralization. MT2 cells are added to a mixture ofvirus (sufficient to result in greater than 80% cell death at 5 dayspost infection) and sera which had been allowed to incubate forapproximately 1 hr. After 5 days in culture, cell viability is measuredby vital dye metabolism. FIG. 6 b shows the percent neutralization foreach bleed at a 1:10 dilution against HIV-1_(MN) in an assay formatemploying CEM targets and p24 endpoint. In this assay, sera areincubated with 200 TCID₅₀ of virus for 1 hr prior to the addition ofcells. On days 1, 3, and 5 media are changed. On day 7 culturesupernatants are collected and analyzed for virus replication by p24antigen levels. In each assay format, percent neutralization isdetermined by comparison of experimental wells with cell and cell/viruscontrols.

The pattern of virus neutralization observed in the previous assays isrepeated. At this serum dilution, bleed #2 for guinea pigs 233 and 234gave 80% and 90% virus neutralization, respectively. The same pattern ofresults was observed against the HIV-1_(SF2) isolate where underidentical assay conditions bleed #2 from animals 233 and 234 gave 70%and 50% neutralization. Control animals receiving adjuvant onlyexhibited no neutralizing activity.

These sera neutralize the HIV-1 isolates MN, RF, and SF2. These resultsindicate a breadth of activity unseen in most other subunit immunogens.By comparison, sera generated against V3 peptides are restricted intheir activity to a small set of very closely related isolates. Due tothe nature of the experiment the low antibody titers are not unexpected.These animals were immunized with free peptide formulated in Freund'sadjuvant. Neither carrier molecules nor accessory proteins were used toenhance the immune response to this molecule. Results from bindingassays indicate low but appreciable levels of antibody against viralenvelope.

In ELISA assays using recombinant gp41 endpoint titers of1:6400-1:44,800 were observed for these samples. Linking P-18 to KLH (orother carrier molecules) and/or administering the conjugate in anadjuvant designed to enhance the immunogenicity of subunit antigens isexpected to result in a significant increase in neutralizing response.

EXAMPLE 3

In a second study, 2 out of 3 animals immunized with P-18 neutralizedthe HIV-1 MN isolate in the assay using the MT2 cell line.

animal neut50 titer BT 004 1:21 BT 005 1:14

Also, one animal receiving P-18 coupled to KLH neutralized the MNisolate in the same assay format.

animal neut50 titer BT 007 1:15

EXAMPLE 4

The peptide used to generate the immune response in Example 2 includeswithin its sequence the linear epitope for the 2F5 monoclonal antibody.To determine if our immune response was against this same region ofenvelope, or involved a previously unidentified neutralizing epitope, aseries of binding experiments was carried out to characterize thereactivity of our polyclonal sera. As can be seen in Table 1, at adilution of 1:100 all animals exhibit good ELISA binding to the cognateimmunogen (P-18). Sera from these animals also have substantial antibodytiters against a peptide derived from the N-terminal P-18 sequence, P1(Table VII). However, when tested at this same dilution against a pairof C-terminal P-18 analogs, P2 and P3 (Table VII) no ELISA reactivitywas observed (Table VIII). This result is significant in that the P3peptide includes the linear binding region (ELDKWAS) for the 2F5monoclonal antibody. These results demonstrate that the neutralizingactivity in our sera is not due to binding to the 2F5 epitope.

TABLE VII Set of three overlapping peptides corresponding to the P-18 peptide P1 YTSLIHSLIEESQNQQEK (SEQ ID NO: 77) P2         EESQNQQEKNEQELLELD (SEQ ID NO: 78) P3                       LELDKWASLWNWF (SEQ ID NO: 79) P-18YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 5)

TABLE VIII ELISA binding at 1:100 (OD) Sample P1 P2 P3 P-18 gp232-20.833 0.124 0.003 1.423 gp232-3 0.858 0.022 0.009 1.067 gp233-2 1.0240.019 0.010 1.314 gp233-3 0.885 0.015 0.015 1.161 gp234-2 0.492 0.0150.016 1.152 gp234-3 0.796 0.012 0.009 0.913

ELISA binding by guinea pig sera to P-18 and a set of overlappingpeptides corresponding to P-18. The majority of the antibody binding isto P-18 and the N-terminal peptide P1. Very little or no reactivity isobserved against P2 and P3 modeling the C-terminal region of P-18.

EXAMPLE 5 Expression of Recombinant gp41 Construct

The plasmid for expression of the construct containing the N- andC-helical domains of HIV-1 gp41 separated by a short linker sequence(See FIG. 7) was prepared as follows. The bacterial expression vectorpTCLE-ssG2C, (based on pAED-4, a T7 expression vector developedspecifically for the expression of small proteins) provided by Dr.Terrance Oas, Duke University was digested at the unique restrictionsites NdeI and EcorI and gel purified using the Qiaex system. The DNAfragments encoding the N- and C-helical regions of HIV-1 gp41 and ashort linker sequence were PCR amplified by standard techniques fromgp41 expression vectors using the following primers.

N-Helix Primer Pair:

(SEQ ID NO: 80) 5′; 5′ GGG CCC ATA TGG GTA TTG TTC AGC AG 3′(includes NdeI site), (SEQ ID NO: 81) 3′; 5′GGG CCG GCG CCT GAG CCG CCG CCT TGA TCC TTC AGG TAG CGT TC 3′(includes NarI site).

C-Helix Primer Pair:

(SEQ ID NO: 82) 5′; 5′ GGG CCG GCG CCG GCT CAG AGT GGG ACAGAG AAA TTA ACA ATT AC 3′ (includes NarI site), (SEQ ID NO: 83) 3′; 5′GGG CCG AAT TCT TAA AAC CAA TTC CAC AAA CTT GCC CAT TT 3′(includes EcorI site and a stop codon).

These fragments were inserted (blunt end ligation) into the TA vectorwhich was amplified to generate larger amounts of DNA. The fragmentscoding for to the N and C-helices were released from the TA vector byrestriction digest (C-helix: NarI and EcoRI, N-helix: NdeI and NarI) andgel purified. A three-way ligation was performed using standardprocedures to introduce the DNA coding for the N- and C-helicalfragments into the pTCLE-ssG2C vector. The product of this step wascharacterized by restriction digestion and DNA sequencing. The vectorcontaining the desired gp41 coding region was prepared in large quantityand BL-21 E. coli host cells were transformed and induced to express thedesired protein. The desired proteins may or may not have a methionineas the first amino acid at he N-terminus. Over-expression of a proteinof the appropriate molecular weight was observed by SDS-Page gelelectrophoresis.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those in the artto which the invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference in theirentirety.

1-14. (canceled)
 15. A method of raising a broadly neutralizing antibodyresponse to HIV, comprising: administering to a mammal a compositionincluding one or more peptides or polypeptides which comprise amino acidsequences that are capable of forming solution stable structures thatcorrespond to, or mimic, the gp41 core six helix bundle.
 16. The methodof claim 15, wherein said one or more peptides or polypeptides comprisea mixture of C-helical peptide or polypeptide and N-helical peptide orpolypeptide.
 17. The method of claim 15, wherein at least one of saidpeptides or polypeptides is multimeric, or is a conjugate structurecomprised of an N-helical domain amino acid sequence and a C-helicaldomain amino acid sequence.
 18. The method of claim 15, wherein saidmixture of C-helical peptide or polypeptide and N-helical peptide orpolypeptide forms a stable core helix solution structure.
 19. The methodof claim 15, wherein said mixture comprises: P-17 and P-18, P-15 andP-16, P-17 and P-16 or P-15 and P-18. 20-32. (canceled)
 33. Acomposition comprising polyclonal or monoclonal antibodies thatspecifically bind to a polypeptide comprising: (a) one or more aminoacid sequences that are capable of forming a stable coiled-coil solutionstructure corresponding to or mimicking the heptad repeat region of gp41(N-helical domain); and (b) one or more amino acid sequences thatcorrespond to, or mimic, an amino acid sequence of thetransmembrane-proximal amphipathic α-helical segment of gp41 (C-helicaldomain); wherein said one or more sequences (a) and (b) are alternatelylinked to one another via a bond, such as a peptide bond (amide linkage)or by an amino acid linking sequence consisting of about 2 to about 25amino acids.
 34. A method of treatment, comprising: administering to anindividual a composition comprising polyclonal or monoclonal antibodiesas claimed in claim 33 in an amount effective to reduce HIV infection ofuninfected cells.
 35. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence encoding a peptide or polypeptide conjugate comprising (a)oneor more amino acid sequences that are capable of forming a stablecoiled-coil solution structure corresponding to or mimicking the heptadrepeat region of gp41 (N-helical domain); and (b) one or more amino acidsequences that correspond to, or mimic, an amino acid sequence of thetransmembrane-proximal amphipathic α-helical segment of gp41 (C-helicaldomain); wherein said one or more sequences (a) and (b) are alternatelylinked to one another via a bond, such as a peptide bond (amide linkage)or by an amino acid linking sequence consisting of about 2 to about 25amino acids.
 36. The nucleic acid molecule of claim 35, wherein saidpolynucleotide has the nucleotide sequence in FIG.
 7. 37. (canceled) 38.A recombinant vector comprising the nucleic acid molecule of claim 35.39. (canceled)
 40. A recombinant host cell comprising the vector ofclaim
 38. 41. A recombinant method for producing a conjugate peptide orpolypeptide, comprising culturing the recombinant host cell of claim 40under conditions such that said polypeptide is expressed and recoveringsaid polypeptide.
 42. The method of claim 15 wherein said administeringis provided in advance of any symptoms of HIV infection, or in advanceof any known exposure to HIV.
 43. The method of claim 15 wherein saidadministering is provided upon or after the detection of symptoms whichindicate that an animal may be infected with HIV, or upon or afterexposure to the virus.